Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway

Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.

Newly Detected Transmission of bla_(KPC-2) by Outer Membrane Vesicles in Klebsiella Pneumoniae

Objective The prevalence of carbapenem-resistant Klebsiella pneumoniae(CR-KP)is a global public health problem.It is mainly caused by the plasmid-carried carbapenemase gene.Outer membrane vesicles(OMVs)contain toxins and other factors involved in various biological processes,includingβ-lactamase and antibiotic-resistance genes.This study aimed to reveal the transmission mechanism of OMV-mediated drug resistance of Klebsiella(K.)pneumoniae.Methods We selected CR-KP producing K.pneumoniae carbapenemase-2(KPC-2)to study whether they can transfer resistance genes through OMVs.The OMVs of CR-KP were obtained by ultracentrifugation,and incubated with carbapenem-sensitive K.pneumoniae for 4 h.Finally,the carbapenem-sensitive K.pneumoniae was tested for the presence of bla_(KPC-2)resistance gene and its sensitivity to carbapenem antibiotics.Results The existence of OMVs was observed by the electron microscopy.The extracted OMVs had bla_(KPC-2)resistance gene.After incubation with OMVs,bla_(KPC-2)resistance gene was detected in sensitive K.pneumoniae,and it became resistant to imipenem and meropenem.Conclusion This study demonstrated that OMVs isolated from KPC-2-producing CR-KP could deliver bla_(KPC-2)to sensitive K.pneumoniae,allowing the bacteria to produce carbapenemase,which may provide a novel target for innovative therapies in combination with conventional antibiotics for treating carbapenem-resistant Enterobacteriaceae.

Proteomic and Functional Analyses of Outer Membrane Vesicles Secreted by Vibrio splendidus

Vibrio splendidus is an important opportunistic pathogen ubiquitously present in the marine environment,exhibiting virulence to a variety of cultured animals.The extracellular products secreted by V.splendidus are crucial to bacterial survival and virulence.In this study,the secretion of outer membrane vesicles(OMVs)by V.splendidus was determined,purified,and morphologically characterized.The protein composition of OMVs was analyzed by proteomic analysis.The results showed that approximately 120 proteins were contained in these OMVs,including outer membrane proteins,flagellins,ABC transporters,protease,and iron regulation proteins,etc.,which were involved in bacterial motility,formation of biofilms and the cell membrane components,and cellular localization based on their structural molecule activity,passive transmembrane transporter activity,channel activity,neurotransmitter receptor activity,extracellular ligand-gated ion channel activity,glutamate receptor activity,ligand-gated ion channel activity,and transmembrane signaling receptor activity.To explore the biological functions of OMVs in V.splendidus,the effects of OMVs on the bacterial adaption to iron limitation,antibiotic,and the coelomic fluid of the Apostichopus japonicus were confirmed.This study is the first time to show that V.splendidus secretes OMVs,and OMVs carry functional proteins that enhance bacterial survival under various stresses.

Membrane tension evolution and mechanical regulation of melittin-induced membrane poration

Membrane tension plays a crucial role in various fundamental cellular processes,with one notable example being the T cell-mediated elimination of tumor cells through perforin-induced membrane perforation by amplifying cellular force.However,the mechanisms governing the regulation of biomolecular activities at the cell interface by membrane tension remain elusive.In this study,we investigated the correlation between membrane tension and poration activity of melittin,a prototypical pore-forming peptide,using dynamic giant unilamellar vesicle leakage assays combined with flickering tension analysis,molecular dynamics simulations,and live cell assays.The results demonstrate that an increase in membrane tension enhances the activity of melittin,particularly near its critical pore-forming concentration.Moreover,peptide actions such as binding,insertion,and aggregation in the membrane further influence the evolution of membrane tension.Live cell experiments reveal that artificially enhancing membrane tension effectively enhances melittin’s ability to induce pore formation and disrupt membranes,resulting in up to a ten-fold increase in A549 cell mortality when exposed to a concentration of 2.0-μg·mL^(-1)melittin.Our findings elucidate the relationship between membrane tension and the mechanism of action as well as pore-forming efficiency of melittin,while providing a practical mechanical approach for regulating functional activity of molecules at the cell-membrane interface.

Phosphatidylglycerol-containing ER-transport vesicles built and restore outer mitochondrial membrane and deliver nuclear DNA translation products to generate cardiolipin in the inner mitochondrial membrane

Phosphatidylglycerol (PG) an important membrane phospholipid required for the synthesis of diphos-phatidylglycerol (DPG) commonly known as cardiolipin (CL) was identified in the fraction of endo-plasmic reticulum (ER)-derived transport vesicles which had no affinity for Golgi. The vesicles were produced in the presence of Brefeldin A (BFA), the agent known to inhibit ER-Golgi transport, and found to display affinity to mitochondria. The analysis revealed that their cargo was not containing proteins that are transported to Golgi, and that their membrane was free of phosphatidylinositol (PI) and ceramides (Cer). The incubation of PG-containing transport vesicles with mitochondria afforded incorporation of their membrane into the Outer Mito-chondrial Membrane (OMM) and formation of lyso-phosphatidylglycerol (LPG). In turn, upon further incubation with fresh transport active cytosol, the mitochondrial LPG was converted to PG. The results of analysis of the OMM, Inner Mitochondrial Mem-brane (IMM) and Inner Mitochondrial Space Components (IMSC) strongly suggest that PG-containing transport vesicles deliver nuclear DNA translation products to the IMSC and thus facilitate CL synthesis in the IMM. In summary, our studies provide evidence that ER-generated PG-enriched transport vesicles represent the general pathway for restitution of mitochondrial membranes and the delivery of nuclear DNA translation products that generate CL, and thus sustain the mitochondrial matrix CL-dependent metabolic reactions.

Gibbs-Donnan Potential as a Tool for Membrane Vesicles Polarization

It has been theoretically predicted that under conditions leading to Gibbs-Donnan equilibrium in case when size of one compartment is very different from another (as in system “membrane vesicle/liposomes—incubation medium”) stable transmembrane potential can be formed, which value is sufficient to fit requirement of real transmembrane potential. Four partial cases were considered with different location and charge of impermeable ion and it was concluded that locations of impermeable ions in medium provide stable transmembrane potential with sufficient value of 60 - 70 mV. Potential-sensitive probe, such as DiOC6(3) and oxonol VI, were used to confirm the calculated potential. According to the change in fluorescence level and emission/excitation shift, a stable and relatively high transmembrane potential can be formed if salt of impermeable ion is located in incubation medium. Impermeable cations and anions may be used to create positive and negative transmembrane potential respectively.

ACTIVE CALCIUM TRANSPORT IN PLASMA MEMBRANE VESICLES FROM DEVELOPING COTYLEDONS OF COMMON BEAN

Plasma membrane vesicles were prepared from the developing cotyledons of common beau (Phaseolus vulgaris L cv Diyundou) by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes.The putative plasma membrane fraction was minimally contaminated by membranes Other than plasma membrane and hence was of high purity. It exhibited a Ca2+dependent ATPase activity, which was inhibited by 1μ mol/L EB and promoted by calcium ionophore A23187. Such an activity was responsible for the observed ATPdependent 45Ca2+ uptake into inside-out plasma membrane vesicles. This process was stimulated by 0.6μmol/L CaM and 20μmol/L IAA but inhibited by 2μmol/L ABA and abolished by A23187. Possible role of cytoplasmic Ca2+ in mediating phytohormones activity is discussed.

不同生长期坏死杆菌OMVs的分离鉴定及其对RAW264.7细胞分泌TNF-α、IL-8的影响

为初步探究坏死杆菌不同生长期外膜囊泡(OMVs)在形态、蛋白组成和功能的差异,利用密度梯度离心法提取坏死杆菌OMVs,通过透射电镜观察OMVs的形态结构,SDS-PAGE和Western blot分析OMVs的蛋白组成差异,ELISA检测OMVs刺激RAW264.7细胞后TNF-α和IL-8的分泌情况。结果显示:坏死杆菌在培养2 h后进入对数生长期,在8 h时达到峰值,随后进入稳定期和衰亡期;坏死杆菌不同生长期均可产生球形的OMVs,在坏死杆菌培养24 h时产生的OMVs中43 K OMP和白细胞毒素的水平显著高于其他各组(P<0.01);坏死杆菌培养12 h时产生的OMVs刺激RAW264.7细胞12 h和24 h后分泌的IL-8和TNF-α水平均高于其他组。因此,坏死杆菌稳定期产生的OMVs可以更好的刺激RAW264.7细胞产生炎症因子IL-8和TNF-α。

Bioengineered exosomal-membrane-camouflaged abiotic nanocarriers: neurodegenerative diseases, tissue engineering and regenerative medicine

A bio-inspired strategy has recently been developed for camouflaging nanocarriers with biomembranes,such as natural cell membranes or subcellular structure-derived membranes.This strategy endows cloaked nanomaterials with improved interfacial properties,superior cell targeting,immune evasion potential,and prolonged duration of systemic circulation.Here,we summarize recent advances in the production and application of exosomal membrane-coated nanomaterials.The structure,properties,and manner in which exosomes communicate with cells are first reviewed.This is followed by a discussion of the types of exosomes and their fabrication methods.We then discuss the applications of biomimetic exosomes and membrane-cloaked nanocarriers in tissue engineering,regenerative medicine,imaging,and the treatment of neurodegenerative diseases.Finally,we appraise the current challenges associated with the clinical translation of biomimetic exosomal membrane-surface-engineered nanovehicles and evaluate the future of this technology.

Dynamic shielding of bacterial outer membrane vesicles for safe and efficient chemo-immunotherapy against tumors

Bacterial outer membrane vesicles(OMVs)are potent immunostimulants of regulating the tumor microenvironment(TME)for immunotherapy,and can be used to deliver drugs.However,the severe systemic inflammatory response triggered by OMVs upon intravenous(i.v.)injection has limited their application.Here,we developed a safe and effective strategy by conjugating doxorubicin-loaded serum albumin(SA-DOX,AD)onto the surface of OMVs using a matrix metalloproteinase(MMP)-cleavable peptide linker(cL).This approach enabled the dynamic shielding of OMVs to reduce the systemic side effects while simultaneously enhancing the anti-tumor effects through chemo-immunotherapy.Specifically,the resulting OMV-cL-AD formulation exhibited significantly enhanced accumulation at the tumor site after i.v.administration,facilitated by the SA decoration on the OMVs surface.Subsequently,the shield on the OMV-cL-AD was cleaved by the over-expressed MMP in the TME,leading to the release of both OMVs and AD.This process provided OMV-induced immunotherapy and DOX-induced chemotherapy,resulting in synergistic tumor inhibition.In conclusion,our work demonstrated the potential of OMV-cL-AD as an effective immunochemotherapy strategy that can prolong the survival time of mice without inducing side effects.

Amniotic membrane mesenchymal stromal cell-derived secretome in the treatment of acute ischemic stroke:A case report

BACKGROUND Stroke is the second and third leading cause of death and disability,respectively.To date,no definitive treatment can repair lost brain function.Recently,various preclinical studies have been reported on mesenchymal stromal cells(MSCs)and their derivatives and their potential as alternative therapies for stroke.CASE SUMMARY A 45-year-old female suffered an acute stroke,which led to paralysis in the left upper and lower limbs.The amniotic membrane MSC-derived secretome(MSCsecretome)was intravenously transplanted once a week for 4 wk.MSC-secretomeregulated regulatory T cells were investigated for the beneficial effects.The clinical improvement of this patient was accompanied by an increased frequency of regulatory T cells after transplantation.CONCLUSION Intravenous administration of MSC-secretome can potentially treat patients who suffer from acute ischemic stroke.

Bacterial outer membrane vesicle-based cancer nanovaccines

Tumor vaccines,a type of personalized tumor immunotherapy,have developed rapidly in recent decades.These vaccines evoke tumor antigen-specific T cells to achieve immune recognition and killing of tumor cells.Because the immunogenicity of tumor antigens alone is insufficient,immune adjuvants and nanocarriers are often required to enhance anti-tumor immune responses.At present,vaccine carrier development often integrates nanocarriers and immune adjuvants.Among them,outer membrane vesicles(OMVs)are receiving increasing attention as a delivery platform for tumor vaccines.OMVs are natural nanovesicles derived from Gramnegative bacteria,which have adjuvant function because they contain pathogen associated molecular patterns.Importantly,OMVs can be functionally modified by genetic engineering of bacteria,thus laying a foundation for applications as a delivery platform for tumor nanovaccines.This review summarizes 5 aspects of recent progress in,and future development of,OMV-based tumor nanovaccines:strain selection,heterogeneity,tumor antigen loading,immunogenicity and safety,and mass production of OMVs.

Bacterial outer membrane vesicles in the fight against cancer

Bacterial outer membrane vesicles(OMVs)are diminutive vesicles naturally released by Gram-negative bacteria.These vesicles possess distinctive characteristics that attract attention for their potential use in drug administration and immunotherapy in cancer treatment.Therapeutic medicines may be delivered via OMVs directly to the tumor sites,thereby minimizing exposure to healthy cells and lowering the risk of systemic toxicity.Furthermore,the activation of the immune system by OMVs has been demonstrated to facilitate the recognition and elimination of cancer cells,which makes them a desirable tool for immunotherapy.They can also be genetically modified to carry specific antigens,immunomodulatory compounds,and small interfering RNAs,enhancing the immune response to cancerous cells and silencing genes associated with disease progression.Combining OMVs with other cancer treatments like chemotherapy and radiation has shown promising synergistic effects.This review highlights the crucial role of bacterial OMVs in cancer,emphasizing their potential as vectors for novel cancer targeted therapies.As researchers delve deeper into the complexities of these vesicles and their interactions with tumors,there is a growing sense of optimism that this avenue of study will bring positive outcomes and renewed hope to cancer patients in the foreseeable future.

Tailoring therapeutics via a systematic beneficial elements comparison between photosynthetic bacteria-derived OMVs and extruded nanovesicles

Photosynthetic bacteria(PSB)has shown significant potential as a drug or drug delivery system owing to their photothermal capabilities and antioxidant properties.Nevertheless,the actualization of their potential is impeded by inherent constraints,including their considerable size,heightened immunogenicity and compromised biosafety.Conquering these obstacles and pursuing more effective solutions remains a top priority.Similar to extracellular vesicles,bacterial outer membrane vesicles(OMVs)have demonstrated a great potential in biomedical applications.OMVs from PSB encapsulate a rich array of bioactive constituents,including proteins,nucleic acids,and lipids inherited from their parent cells.Consequently,they emerge as a promising and practical alternative.Unfortunately,OMVs have suffered from low yield and inconsistent particle sizes.In response,bacteria-derived nanovesicles(BNVs),created through controlled extrusion,adeptly overcome the challenges associated with OMVs.However,the differences,both in composition and subsequent biological effects,between OMVs and BNVs remain enigmatic.In a groundbreaking endeavor,our study meticulously cultivates PSB-derived OMVs and BNVs,dissecting their nuances.Despite minimal differences in morphology and size between PSB-derived OMVs and BNVs,the latter contains a higher concentration of active ingredients and metabolites.Particularly noteworthy is the elevated levels of lysophosphatidylcholine(LPC)found in BNVs,known for its ability to enhance cell proliferation and initiate downstream signaling pathways that promote angiogenesis and epithelialization.Importantly,our results indicate that BNVs can accelerate wound closure more effectively by orchestrating a harmonious balance of cell proliferation and migration within NIH-3T3 cells,while also activating the EGFR/AKT/PI3K pathway.In contrast,OMVs have a pronounced aptitude in anti-cancer efforts,driving macrophages toward the M1 phenotype and promoting the release of inflammatory cytokines.Thus,our findings not only provide a promising methodological framework but also establish a definitive criterion for discerning the optimal application of OMVs and BNVs in addressing a wide range of medical conditions.

Suppression of host plant defense by bacterial small RNAs packaged in outer membrane vesicles

Noncoding small RNAs(sRNAs)packaged in bacterial outer membrane vesicles(OMVs)function as novel mediators of interspecies communication.While the role of bacterial sRNAs in enhancing virulence is well established,the role of sRNAs in the interaction between OMVs from phytopathogenic bacteria and their host plants remains unclear.In this study,we employ RNA sequencing to characterize differentially pack-aged sRNAs in OMVs of the phytopathogen Xanthomonas oryzae pv.oryzicola(Xoc).Our candidate sRNA(Xosr001)was abundant in OMVs and involved in the regulation of OsJMT1 to impair host stomatal immu-nity.Xoc loads Xosr001 into OMVs,which are specifically ttransferred into the mechanical tissues of rice leaves.Xosr001 suppresses OsJMT1 transcript accumulation in vivo,leading to a reduction in MeJA accu-mulation in rice leaves.Furthermore,the application of synthesized Xosr001 sRNA to the leaves of OsJMT1-HA-OE transgenic line results in the suppression of OsJMT1 expression by Xosr001.Notably,the OsJMT1-HA-OE transgenic line exhibited attenuated stomatal immunity and disease susceptibility upon infection with DXosr001 compared to Xoc.These results suggest that Xosr001 packaged in Xoc OMVs functions to suppress stomatal immunity in rice.

CD133 and membrane microdomains:Old facets for future hypotheses

Understanding all facets of membrane microdomains in normal and cancerous cells within the digestive tract is highly important,not only from a clinical point of view,but also in terms of our basic knowledge of cellular transformation.By studying the normal and cancer stem cell-associated molecule CD133 (prominin-1),novel aspects of the organization and dynamics of polarized epithelial cells have been revealed during the last decade.Its association with particular membrane microdomains is highly relevant in these contexts and might also offer new avenues in diagnosis and/or targeting of cancer stem cells.

The Flow of Information from Nucleus to Golgi Is Contingent upon Nuclear Membrane Synthesis and Protraction of the Ceramide-Containing Membrane to Endoplasmic Reticulum

The nucleus-initiated augmentation of ER membrane is reflected in a coordinated synthesis and intercalation of the explicit proteins and lipids required for the replacement, repair and function of the cell and its organelles. The direct connection between nucleus and the membranes containing labeled sphingosine (SphN) and ceramide (Cer) was affirmed by determining synthetic activity of serine palmitoyltransferase (SPT). The SPT and the newly synthesized serine-labeled lipid products were identified in the Outer- and Inner-Nuclear Membrane (ONM, INM) and ER. The pulse-chase experiments disclosed that the incorporation of radiolabeled lipids into both nuclear membranes declined upon their simultaneous increase in Endoplasmic Reticulum (ER). These results, and prior findings regarding metabolic transfer of nuclear membrane phosphoinositides to the outer leaflet of ER [Slomiany and Slomiany, Health, 2011, 3, 187-199], allowed us to reason that INM and ONM are not distinct entities, but uninterrupted continuum facing nucleosol and then cytosol when protracted into segment known as ER. Consequently, the identification of SPT and its products in the inner leaflet of nuclear and ER microsomes lent credence to the luminal presence of Cer in Golgi, luminal synthesis of glycosphingolipids (GSphLs), sphingomyelin (SM), and their delivery to the outer leaflet of apical and basolateral cell membrane, respectively. The findings presented in this communication provide further support to our concept that the factual intercalation of proteins and lipids into the cell membranes can only take place during their simultaneous synthesis that is guided by the nuclear and cytosolic processes enacted in nuclear-ER membrane continuum. At the nuclear stage, the signal-specific genes expression promotes active synthesis and intercalation of lipids into the organelles’ customized membrane that is protracted and articulated in ER in form of transport vesicles.

Mitochondrial Membranes Restitution Proceeds via Vesicular Import from ER and Cytosol. Counterparts’ Resemblances and Variances in Mitochondria and Golgi Pathways

The processes of mitochondrial restitution are controlled by nuclear genes that encode proteins synthesized in ER and cytosol and delivered as organelle- and membrane-specific transport vesicles. The analysis of the transporters recovered from inner mitochondrial space (Mitosol) revealed that the ER-synthesized mitochondria-specific transport vesicles consist of two carriers, one remaining in outer mitochondrial membrane (OMM), and the other that transfers specific membrane segments to the inner mitochondrial membrane (IMM). The ER-assembled and IMM-committed membrane segments, while first integrated into OMM, undergo intra-mitochondrial lipid modification reflected in the synthesis of cardiolipin (CL) and inversion into Mitosol with load of IMM associated cytosolic proteins. Then, the CL-bedecked vesicles are released from OMM to Mitosol and upon contact with IMM fuse with the membrane, and the release of cytosolic cargo ensues. While ER-assembled mitochondria-specific transport vesicles fuse with OMM with the aid of the cytosolic, phosphatidylglycerol (PG)-specific phospholipase A2 (PLA2), the Mitosol-contained CL-specific PLA guides vesicles fusion with IMM. The described path of translocation of the membrane segments and the cytosol synthesized proteins into the designated mitochondrial compartments sustains growth and identity of OMM, IMM, maintains protein delivery for intra-mitochondrial lipid and protein modification in Mitosol, and ensures conformity of the cytosolic proteins cargo delivered to matrix.

Bacterial outer membrane vesicles-based therapeutic platform eradicates triple-negative breast tumor by combinational photodynamic/chemo-/immunotherapy

Bacterial outer membrane vesicles(OMVs)are potent immuno-stimulating agents and have the potentials to be bioengineered as platforms for antitumor nanomedicine.In this study,OMVs are demonstrated as promising antitumor therapeutics.OMVs can lead to beneficial M2-to-M1 polarization of macrophages and induce pyroptosis to enhance antitumor immunity,but the therapeutic window of OMVs is narrow for its toxicity.We propose a bioengineering strategy to enhance the tumor-targeting ability of OMVs by macrophage-mediated delivery and improve the antitumor efficacy by co-loading of photosensitizer chlorin e6(Ce6)and chemotherapeutic drug doxorubicin(DOX)into OMVs as a therapeutic platform.We demonstrate that systemic injection of the DOX/Ce6-OMVs@M therapeutic platform,providing combinational photodynamic/chemo-/immunotherapy,eradicates triple-negative breast tumors in mice without side effects.Importantly,this strategy also effectively prevents tumor metastasis to the lung.This OMVs-based strategy with bioengineering may serve as a powerful therapeutic platform for a synergic antitumor therapy.

Transformation of Structure and Properties of Vesicles Induced by Transition Metal Ions

This paper proved that octodecyl propylenediamine could form vesicles in pure water and aqueous solution of CuCl2 or Cu（NO3）2. The structure and morphology of vesicles were different when the copper （Ⅱ） salt was added to the solution. The results showed that both the counterions and the ligands had strong influence on the configuration of coordinated structures and packing model in bilayer membrane of vesicles.